Bovine Chromaffin Cells Release a Transforming Growth Factor-β-Like Molecule Contained Within Chromaffin Granules

Abstract: Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)-β in terms of its biological activity. The assay used to assess the activity of TGF-β is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-β1, -β2, and -β3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-α, platelet-derived growth factor-AB, insulin-like growth factor-I, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-β superfamily members (activin A, bone morphogenetic protein-2, -4, -6, and -7, growth/differentiation factor-5, and glial cell line-derived neurotrophic factor). Chromaffin granules contain ∼1 ng of TGF-β/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-β1/β2/β3. TGF-β is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10⁻⁵ M ). These data suggest that TGF-β is stored in chromaffin granules and can be released by exocytosis.     

FGF modulates the PDGF-driven pathway of oligodendrocyte development

PDGF promotes the growth of oligodendrocyte type-2 astrocyte (O-2A) glial progenitor cells and allows their timely differentiation into oligodendrocytes, the CNS myelin-forming cells. We demonstrate that basic FGF is a potent mitogen for brain O-2A progenitor cells, but blocks their differentiation into oligodendrocytes. Treatment with basic FGF also influences the level of expression of PDGF receptors on O-2A progenitor cells. These cells express only the alpha chain PDGF receptor, and the levels of PDGF alpha receptors decrease as the cells differentiate. In contrast, basic FGF maintains a high level of functionally responsive PDGF alpha receptors in O-2A progenitors. Thus basic FGF activates a signaling pathway that can positively regulate PDGF receptors in O-2A progenitor cells. In this way basic FGF or an FGF-like factor may modulate the production of myelin-forming cells in the CNS.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.     

Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.     

IGF-I synergizes with FGF-2 to stimulate oligodendrocyte progenitor entry into the cell cycle

Secreted peptide growth factors are critical extracellular signals that interact to promote the proliferation, differentiation, and survival of progenitor cells in developing tissues. IGF-I signaling through the IGF type I receptor provides a mitogenic signal for numerous cell types, including stem and progenitor cells. We have utilized the O-2A oligodendrocyte progenitor to study the mechanism of IGF-I mitogenic actions since these progenitors respond to IGF-I in vitro, and gene targeting studies in mice have demonstrated that IGF-I is essential for normal oligodendrocyte development in vivo. The goal of this study was to elucidate the mechanism by which IGF-I promotes the proliferation of oligodendrocyte progenitors in the context of other mitogens critical for their proliferation. Results presented here show that IGF-I significantly amplified the actions of FGF-2 and PDGF to promote DNA synthesis in O-2A progenitors. Investigation of cell cycle kinetics revealed that IGF-I had no significant effect on the rate of cell cycle progression. Instead, IGF-I promoted increased recruitment of O-2A progenitors into the S phase of the cell cycle. These studies support a role for IGF-I as a cell cycle progression factor for progenitor cells.     

Isoform-Specific Effects of Transforming Growth Factors-β on Degeneration of Primary Neuronal Cultures Induced by Cytotoxic Hypoxia or Glutamate

Abstract: The transforming growth factors-β (TGFs-β) are multifunctional peptide-growth factors that have been localized in neuronal and glial cells of the C'NS of mice. rats, and chick embryos. We tested the TGF-β isoforms 1. 2. and 3 for their protective ctlects against neuronal degeneration caused by cytotoxic hypoxia or by the excitatory amino acid i -glutamate. A cytotoxic hypoxia was induced in cultured chick embryo telencephalic neurons by adding I m.l/ sodium cyanide to the culture medium tor a period of 30min. I reatment with TGF-81 (1-30 ng/ml) led to a statistically significant increase in cell viability, neuronal ATP levels. and protein content of the cultures assessed 72 h after the toxic insult. TGF-33 was able to reduce the cyanide-induced neuronal damage at concentrations of 0.3 and 1 ng/ ml. whereas TGF-33, only showed neuroprotective activity at concentrations of 30 and 50 ng/ml. Both pre- and posttreatment with TGF-31, also prevented the degeneration of cultured chick embryo telencephalic neurons that had been exposed to I mM L-glutamate in a buffered salt solution for a period of 60 min. Furthermore, TGF-β1 (0.3-3 ng/ml). and to a lesser extent TGF-β3 (0.1-1 ng/ml). significantly reduced excitotoxic injury of cultured neurons from rat cerebral cortex that had been exposed to serum-free culture medium supplemented with 1 m.M L-glutamate. These results demonstrate that the TGFs-β are able to prevent the degeneration of primary neuronal cultures, which was caused by energy depletion and activation of glutamate receptors, in an isoform-specific manner.     

A role for platelet-derived growth factor in normal gliogenesis in the central nervous system

The bipotential progenitor cells (O-2A progenitors) that produce oligodendrocytes and type-2 astrocytes in the developing rat optic nerve are induced to proliferate in culture by type-1 astrocytes. Here, we show that the astrocyte-derived mitogen is platelet-derived growth factor (PDGF). PDGF is a potent mitogen for O-2A progenitor cells in vitro. Mitogenic activity in astrocyte-conditioned medium comigrates with PDGF on a size-exclusion column, competes with PDGF for receptors, and is neutralized by antibodies to PDGF. PDGF dimers can be immunoprecipitated from astrocyte-conditioned medium, and mRNA encoding PDGF is present in rat brain throughout gliogenesis. We propose that astrocyte-derived PDGF is crucial for the control of myelination in the developing central nervous system.     

Bioactive TGF-β can associate with lipoproteins and is enriched in those containing apolipoprotein E3

Transforming growth factor-β1 (TGF-β1) has central functions in development, tissue maintenance, and repair and has been implicated in major diseases. We discovered that TGF-β1 contains several amphipathic helices and hydrophobic domains similar to apolipoprotein E (apoE), a protein involved in lipoprotein metabolism. Indeed, TGF-β1 associates with lipoproteins isolated from human plasma, cultured liver cells, or astrocytes, and its bioactivity was highest in high-density lipoprotein preparations. Importantly, lipoproteins containing the apoE3 isoform had higher TGF-β levels and bioactivity than those containing apoE4, a major genetic risk factor for atherosclerosis and Alzheimer's disease. Because TGF-β1 can be protective in these diseases an association with apoE3 may be beneficial. Association of TGF-β with different types of lipoproteins may facilitate its diffusion, regulate signaling, and offer additional specificity for this important growth factor.     

Postinjury Changes in Platelet-Derived Growth Factor-Like Activity in Fish and Rat Optic Nerves

The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet-derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF-responsive cells. The results suggest that PDGF-like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF-like levels. The results are discussed with respect to the possible role of PDGF in regeneration.     

Growth factor regulation of proliferation in primary cultures of small intestinal epithelium

Summary Although the intestinal epithelium is one of the most rapidly renewing tissues, little is known about the major growth factors that control the rate of cell replacement and migration. Recently, a primary culture model has been described for the developing rat small intestinal epithelium, which permits epithelial growth while maintaining interactions with associated stromal cells, thereby possessing several contextual advantages over established cell lines (Evans et al., 1992). We have used this model to begin to determine the factors that may be involved in controlling intestinal epithelial cell proliferation. Under the conditions examined, no single growth factor promoted exclusive proliferation of epithelial cells; stromal cell proliferation was also apparent. The most potent stimulators of epithelial proliferation were insulin and insulin-like growth factor 1 (IGF-1). These factors also appeared to inhibit migration of the epithelial cells. 5–10 ng/ml EGF, 5–20 ng/ml TGFα, and 10–20 ng/ml PDGF also slightly increased epithelial cell numbers. Cell proliferation was inhibited by 0.1 ng/ml TGFβ-1. In Dulbecco’s modified Eagle’s medium (DMEM) containing 0.25 IU/ml insulin, glucose levels of 2–3 g/liter permitted epithelial growth with limited expansion of the stromal cell population. Higher levels of glucose further stimulated the nonepithelial cell types. Transferrin was also a potent stimulator of both cell types.     

Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Growth, Receptor Phosphorylation, and Dimerization in Neuroblastoma SH-SY5Y Cells

Abstract: SH-SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK-N-SH. It grows well in serum-containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet-derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF-BB, we found that SH-SY5Y cells specifically bind PDGF with a K _D = 0.14 ± 0.06 n M and B _max = 7.3 ± 2.3 p M . Functionality of these receptors was demonstrated by an increased [³H]-thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF-BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12–24-h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [³H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170-kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti-phosphotyrosine antibody. Immunoprecipitation with anti-PDGF β-receptor antibody and visualization on a western blot with an anti-phosphotyrosine antibody also revealed a 170-kDa protein. Maximum phosphorylation of the 170-kDa protein occurred after 5-min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH-SY5Y cells have specific receptors for PDGF-BB that are functional, and can be modulated by gangliosides.     

PDGF and intracellular signaling in the timing of oligodendrocyte differentiation

In the rat optic nerve, bipotential O-2A progenitor cells give rise to oligodendrocytes and type 2 astrocytes on a precise schedule. Previous studies suggest that PDGF plays an important part in timing oligodendrocyte development by stimulating O-2A progenitor cells to proliferate until they become mitotically unresponsive to PDGF, stop dividing, and differentiate automatically into oligodendrocytes. Since the loss of mitotic responsiveness to PDGF has been shown not to be due to a loss of PDGF receptors, we have now examined the possibility that the unresponsiveness results from an uncoupling of these receptors from early intracellular signaling pathways. We show that (a) although PDGF does not stimulate newly formed oligodendrocytes to synthesize DNA, it induces an increase in cytosolic Ca2+ in these cells; (b) a combination of a Ca2+ ionophore plus a phorbol ester mimics the effect of PDGF, both in stimulating O-2A progenitor cell division and in reconstituting the normal timing of oligodendrocyte differentiation in culture; and (c) the same combination of drugs does not stimulate newly formed oligodendrocytes to proliferate, even in the presence of PDGF or dibutyryl cAMP. The most parsimonious explanation for these results is that O-2A progenitor cells become mitotically unresponsive to PDGF because the intracellular signaling pathways from the PDGF receptor to the nucleus are blocked downstream from the receptor and some of the early events that are triggered by receptor activation.     

血小板来源的生长因子通过ERK/p38/PI3K促进人视网膜色素上皮细胞的增殖、迁移

目的:探讨血小板来源的生长因子(PDGF)对体外培养的人视网膜色素上皮细胞(RPE)增殖和迁移的影响,并对参与其中的信号通路做初步研究.方法:体外培养的人视网膜色素上皮细胞与含有重组人血小板来源的生长因子的培养基(含有或不含2％(v/v)胎牛血清)共培养,用MTT法检测PDGF对RPE细胞增殖的影响,利用细胞爬片和免疫荧光技术检测PDGF对RPE细胞迁移等影响;另外分别向细胞培养物中添加PD98059,SB203580和PI3K等不同的信号通路分子抑制剂,判断参与PDGF激活的细胞活动相关的信号通路.结果:外源性PDGF能促进体外培养的人RPE的增殖和迁移.ERK1/2选择性抑制剂PD98059和PI3K抑制剂LY294002能显著的降低PDGF-BB诱导的人RPE细胞的增殖(P＜0.05),p38抑制剂SB203580没有明显的抑制作用.而对PDGF-BB诱导的RPE细胞的迁移,SB203580和LY294002有显著的抑制作用(P＜0.05),PD98059抑制作用不显著.结论:PDGF对RPE细胞的影响提示其在增生性玻璃体视网膜病变(PVR)的发展中有重要的作用,其可能为PVR提供一种新的毒副作用小的治疗手段.